The present R03 resubmission is motivated by progress made by the PI (Piermarini) during the first two years of his K01 award under the mentorship of Prof. Klaus W. Beyenbach at Cornell University. The proposed research is an extension of the PI's on-going molecular and physiological studies of the renal (Malpighian) tubules of the mosquito Aedes aegypti, which are a valuable model for studying transepithelial transport that is powered exclusively by the vacuolar (V)-type H+ATPase. The proposed R03 research will enhance the PI's career development by allowing him to 1) maintain the momentum of his on-going K01 research on Aedes Malpighian tubules, and 2) collect preliminary data for a subsequent R01 application. The proposed studies will also provide insights into mammalian cells that are energized by the V-type H+ATPase. Such insights are of significant biomedical interest, because several important pathologies are now known to be associated with defects or dysfunction of the V-type H+ATPase. Lastly, the proposed research has the potential to 1) shift the current paradigm of renal function in mosquitoes, which are important vectors of diseases that afflict humans, and 2) provide the first example of gap junction- mediated (intercellular) regulation of intracellular pH (pHi) in a renal epithelium. The research goal of the parent K01 award is to decipher the molecular mechanisms of acid-base transport in the 'principal'cells of Aedes Malpighian tubules. These cells resemble acid-secreting (a) intercalated cells of the mammalian renal collecting tubule in that they are mitochondrion-rich and express an apical V-type H+ ATPase. Based on these similarities, the PI hypothesized that principal cells express a basolateral Cl/HCO3 anion exchanger (AE), as is found in mammalian a-intercalated cells. The PI has now shown that the Aedes AE (AeAE) is expressed in the basolateral membrane of the neighboring 'stellate'cells, which intercalate between principal cells. Furthermore, inhibiting AeAE in Malpighian tubules disrupts transepithelial transport in a manner consistent with inhibition of the V-type H+ATPase in principal cells. Thus, the activity of AeAE in stellate cells appears to be coupled to the apical V-type H+ATPase in principal cells. The goal of the present R03 grant application is to determine if gap junctions mediate the putative functional coupling between principal and stellate cells. In aim 1, I will use a pharmacological approach to test the hypothesis that the 1) activity of the V-type H+ATPase in principal cells, 2) regulation of pHi in principal cells, and 3) rates of transepithelial fluid secretion mediated by principal cells, are functionally coupled to the AeAE in stellate cells via gap junctions. In aim 2, I will use a functional genetic approach (RNA interference) to test the hypothesis that innexins-the gap-junction proteins of insects-are the molecular entities that couple principal cells to stellate cells. PUBLIC HEALTH RELEVANCE: The goal of the proposed study is to elucidate a novel mechanism for regulating the activity of a vacuolar-type H+ATPase (proton pump) in the 'kidney'of the mosquito. The mosquito 'kidney'offers a natural (non-cell culture) and accessible model system for studying the function and regulation of the proton pump. This pump is of vital importance to human health, because its dysfunction is associated with important diseases related to impaired acid secretion by the kidney, osteoporosis, deafness, and sterility (in males).